Glut4-mediated glucose uptake represents the rate-limiting step of insulin-stimulated glucose disposal, and type 2 diabetes is associated with impaired translocation of Glut4 to the cell surface. The insulin-responsive pool of Glut4 is localized in intracellular membrane vesicles, or IRVs, that deliver Glut4 to the plasma membrane. Proteomics analysis performed in our and other laboratories have shown that, in addition to Glut4, these vesicles contain two major proteins, IRAP and sortilin. The challenge now is to understand how these seemingly unrelated proteins find each other in the cell and form a unique type of a vesicular carrier. Based on the results obtained in the previous funding period, we put forward a hypothesis that may explain the process of the IRV formation. According to our model, the cytoplasmic tails target the major IRV proteins to the perinuclear donor membranes, where Glut4 and IRAP interact with sortilin via lumenal domains. The heteromeric complex consisting of Glut4, IRAP and sortilin is distributed from the donor membranes to the IRV as a single entity with the help of GGA and ACAP-1 adaptors. In Specific Aim 1, we will test this hypothesis and further characterize the molecular mechanisms of the IRV biogenesis. In Specific Aim 2, we will determine which of the IRV component proteins is responsible for their insulin sensitivity. Recent studies have demonstrated that all the known component proteins of the IRVs, including Glut4, are expressed in the brain at significant levels with cerebellum being the region with the highest Glut4 content. We have found that Glut4 in cerebellum neurons is localized primarily in intracellular vesicles that resemble the IRVs from fat and skeletal muscle cells and are translocated to the plasma membrane in response to insulin stimulation and exercise. In Specific Aim 3, we will characterize the significance of Glut4-mediated glucose uptake in the cerebellum under various experimental conditions in vivo.